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1.
Cardamom is an important spice, condiment and medicine, and international commodity. DNA-based molecular profiling will be
aid in protecting the intellectual property rights of those who trade cardamom on the world market. Commercial cardamom has
so far proven recalcitrant to traditional DNA extraction methods. In this paper we report a protocol for the isolation of
amplifiable genomic DNA from traded cardamom. The method involves a modified CTAB (hexadecyltrimethylammonium bromide) extraction
step, followed by a purification step to remove polysaccharides, proteins, and polyphenols, which are abundant in storage
tissue such as cardamom capsules. The yield of DNA was 6–7 μg g−1 tissue. Spectrophotometric and electrophoretic analysis indicated that the isolated DNA was highly pure and of high molecular
weight. The isolated DNA could be amplified using different random decamer primers. The protocol has trade implications as
it will help in the PCR-based characterisation of traded cardamom. This protocol can be further extended to develop Sequence
Characterised Amplified Regions (SCAR) markers for profiling cardamoms. 相似文献
2.
An aminopeptidase from the mycelia of Streptomyces rimosus was isolated in an electrophoretically homogeneous form. It was shown to be a monomeric, acidic protein (pI = 4.4, mol. wt.
approx. 83,000), with optimal activity at pH 7.1–7.8 and at 35–41° C. The enzyme was fully inhibited by 0.1 mM EDTA or 1 mM
o-phenanthroline; the activity was restored upon addition of 0.05 mM Co2+, Zn2+, or Ni2+. Amastatin, bestatin, and puromycin also inhibited the enzyme. The aminopeptidase hydrolyzed amino-acid-2-naphthylamides
and various di- to heptapeptides. The highest catalytic coefficients (23 and 19 μM–1 s–1) were obtained with Arg- and Lys-2-naphthylamide, followed by Leu-, Phe- and Met-derivatives with one order of magnitude
lower catalytic coefficients. Basic or bulky hydrophobic amino acids at the P1 and/or P1′ position of peptide substrates were preferred. Acidic amino acids and proline were not accepted. The affinity of the enzyme
increased with the length of peptide. According to these properties, S. rimosus intracellular aminopeptidase is distinct from the extracellular leucine aminopeptidase of the same organism and can be classified
as an Arg(Lys)-preferring metalloaminopeptidase.
Received: 18 January 1996 / Accepted: 19 March 1996 相似文献
3.
Abolghasem Abbasi Kejani Sayed Ali Hosseini Tafreshi Sayed Mojtaba Khayyam Nekouei Mohammad Reza Mofid 《Molecular biology reports》2010,37(2):797-800
Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew
(Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and β-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation
were washed only with Chloroform: isoamyl alcohol (24:1). So, we could successfully eliminate the dangerous phenol/chloroform
extraction steps from the isolation procedure. Both spectrophotometric (A260/A280 and A260/A230 ratios) and agarose electrophoresis analysis of isolated nucleic acids (DNA and RNA) indicated good results. DNA with the
average yield of 100–300 μg/g leaf and stem tissue and total RNA with an average yield of 20–30 μg/g cell culture and 80–100 μg/g
leaf and stem tissue of Iranian yew could be obtained. Successful amplification of pam and pds by PCR and RT-PCR, showed the integrity of isolated DNA and RNA, respectively. 相似文献
4.
High-quality plant DNA extraction for PCR: an easy approach 总被引:1,自引:0,他引:1
I. Ahmed M. Islam W. Arshad A. Mannan W. Ahmad B. Mirza 《Journal of applied genetics》2009,50(2):105-107
Polymerase chain reaction has found wide applications in modern research involving transformations and other genomic studies.
For reproducible PCR results, however, the quantity and quality of template DNA is of considerable importance. A simple and
efficient plant DNA extraction procedure for isolation of high-quality DNA from plant tissues is presented here. It requires
maceration of plant tissue of about 1.0 cm2 (e.g. of a leaf blade) in DNA extraction buffer (100 mM Tris-HCl, 100 mM EDTA, 250 mM NaCl) using 1.5-mL microfuge tubes,
followed by cell lysis with 20% SDS, and DNA extraction with phenol: chloroform: iso-amyl alcohol (25:24:1). Hydrated ether
is then used to remove polysaccharides and other contaminants from the DNA preparation. Average DNA yield is 20–30 μg cm−2 for fresh tissues, and ratio of absorbance at 260 nm to absorbance at 280 nm is 1.5–1.8. The DNA is quite suitable for PCR
using microsatellites, RAPD and specific markers for recombinant selection. Amplifications have been obtained for these markers
by using template DNA extracted from fresh as well as frozen leaf tissues of various plants, including barley, oat, potato
and tomato. DNA stored for more than 2 years has been successfully amplified with microsatellite markers, which shows suitability
of this method after long-term storage of DNA. Besides, the ease of use and cost-effectiveness make the procedure attractive. 相似文献
5.
A qualitative procedure of purified DNA/RNA co-extraction from complex organic matter, used as biofilter support for removing
volatile organic compounds, was set up and applied to detect xylene monooxygenase gene expression by RT-PCR. A DNA/RNA extraction
protocol based on a combination of sample lyophilization pre-treatment and CTAB––phenol/chloroform extraction procedure was
optimized for the recovery of purified nucleic acids [100–500 ng DNA (10 kb) and 0.5–2 μg of rRNA 16S from 100 mg matrix].
PCR and RT-PCR protocols were established to detect xylene monooxygenase gene expression starting from differentially induced
organic matrices obtained by biofiltration technology. This work allowed the microbial degradation activities in heterogeneous
organic solid media to be studied and suggests a rapid method to follow specific biological activities during solid and/or
semisolid organic substrates biotransformation. 相似文献
6.
Responses of mycelia ofGanoderma lucidum to vanadium, selenium and germanium were examined over a wide range of concentrations (10–1, 120 μg/ml) in pure culture.
Se and V were found to be highly toxic, but Ge was not toxic at the levels tested.Ganododerma lucidum cultivated on substrates of sawdust with V (30–80 μg/g) developed mature fruitbodies, but the bioaccumulation of V was quite
low (2.5–7 μg/g in pileus, 12.5–21.5 μg/g in stipe and <1 μg/g in basidiospores). Se as Na2SeO4 labeled with75Se was effectively taken up from substrates and accumulated in fruitbodies (mainly in pileus), then depleted by discharge
of basidiospores. Ge as GeCl4 labeled with77Ge was easily uptaken and translocated into fruitbodies. 相似文献
7.
T. I. Odintsova I. M. Vassilieva T. V. Korostyleva L. L. Utkina A. A. Slavokhotova E. A. Rogozhin A. N. Shiyan V. A. Pukhalskii G. D. Zasukhina 《Russian Journal of Genetics》2011,47(9):1128-1131
Antimutagenic activity on human RD cells was studied for β-purothionin Tk-AMP-BP isolated from seeds of wheat Triticum kiharae, which has high stress resistance. Cadmium chloride at 5 × 10−6 M was used as a mutagen. The numbers of DNA breaks in mutagen-treated and intact cells were inferred from the single-stranded
to double-stranded DNA ratio and expressed as protection coefficients. The protective effect was simultaneously assayed for
aqueous plant extracts known to possess antioxidant properties. Wheat thionin was the most active among all of the antimutagens
examined; its protection coefficient reached 85–88% at micromolar peptide concentrations (8–32 μg/ml). Thus, wheat β-purothionin
was for the first time demonstrated to be highly efficient in protecting human cell DNA from the damaging effect of cadmium
chloride. 相似文献
8.
9.
10.
Nevinsky GA 《Biochemistry. Biokhimii?a》2011,76(1):94-117
Specific and nonspecific DNA complex formation with human uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, and apurine/apyrimidine
endonuclease, as well as with E. coli 8-oxoguanine-DNA glycosylase and RecA protein was analyzed using the method of stepwise increase in DNA-ligand complexity.
It is shown that high affinity of these enzymes to any DNA (10−4–10−8 M) is provided by a large number of weak additive contacts mainly with DNA internucleoside phosphate groups and in a less
degree with bases of nucleotide links “covered” by protein globules. Enzyme interactions with specific DNA links are comparable
in efficiency with weak unspecific contacts and provide only for one-two orders of affinity (10−1–10−2 M), but these contacts are extremely important at stages of DNA and enzyme structural adaptation and catalysis proper. Only
in the case of specific DNA individual for each enzyme alterations in DNA structure provide for efficient adjustment of reacting
enzyme atoms and DNA orbitals with accuracy up to 10–15° and, as a result, for high reaction rate. Upon transition from nonspecific
to specific DNA, reaction rate (k
cat) increases by 4–8 orders of magnitude. Thus, stages of DNA and enzyme structural adaptation as well as catalysis proper are
the basis of specificity of repair enzymes. 相似文献
11.
Thermomonospora curvata produced a thermostable β-xylosidase during growth on birch xylan. The enzyme, extracted by sonication of early stationary
phase mycelia, was purified by isoelectric focusing and size exclusion HPLC. The isoelectric point was pH 4.8. The molecular
weight was estimated to be 102 000 by size exclusion HPLC and 112 000 by SDS-PAGE. Maximal activity occurred at pH 6–7 and
60–68°C. K
m values for xylobiose and p-nitrophenyl-β -D-xylopyranoside were 4.0 M and 0.6 M respectively. The enzyme was sensitive to low levels of Hg2+ (50% inhibition at 0.2 μM), but was stimulated by Co2+ and Pb2+. Addition of the xylosidase to a xylanase reaction mixture increased the liberation of xylose equivalents from xylan and
decreased the proportion of xylobiose in the hydrolysate.
Received 14 April 1997/ Accepted in revised form 21 October 1997 相似文献
12.
A modified mini-prep method for economical and rapid extraction of genomic DNA in plants 总被引:1,自引:0,他引:1
Molecular markers for map-based cloning, marker-assisted selection in crop breeding, and genetic studies require DNA isolation
from a large number of plants in a short span of time. Here we describe a modified DNA extraction method that is economical
in terms of cost, time and labour. The method allows DNA extraction from as little as 0.2–0.3 g of leaves that are homogenized
in zipper plastic bags, followed by DNA isolation in 1.5-mL Eppendorf tubes. By using the modified method, a DNA yield of
700–800 μg/300 mg leaf tissue was obtained from cotton and wheat samples. The quality of the DNA was quite suitable for PCR-based
markers. 相似文献
13.
During the course of our research on new actinobacterial sources, a novel actinomycete strain YIM 63101T was isolated from the surface-sterilized roots of Artemisia annua L. collected from Xishuangbanna, Yunnan province, south-west China and characterized by using a polyphasic approach. The
strain formed well-differentiated aerial and substrate mycelia. Phylogenetic analysis based on 16S rRNA gene sequences showed
that strain YIM 63101T belongs to the genus Pseudonocardia, with highest similarity to “Pseudonocardia artemisiae YIM 63587T” (99.4%). Sequence similarities between strain YIM 63101T and the other Pseudonocardia species ranged from 97.0 (Pseudonocardia saturnea IMSNU 20052T) to 94.0% (Pseudonocardia compacta IMSNU 20111T). The chemotaxonomic characteristics, such as cell wall diaminopimelic acid, whole-cell sugars, fatty acid components and
the major menaquinones suggested that the organism belonged to the genus Pseudonocardia. The G + C content of the genomic DNA was 69.4 mol%. Based on comparative analysis of physiological, biochemical and chemotaxonomic
data, including low DNA–DNA hybridization results, it is proposed that strain YIM 63101T represents a novel species of the genus Pseudonocardia, named Pseudonocardia bannaensis sp. nov. The type strain is YIM 63101T (= CCTCC AA 208077 T = DSM 45300T). 相似文献
14.
Yi-Guang Chen Shu-Kun Tang Yu-Qin Zhang Zhu-Xiang Liu Qi-Hui Chen Jian-Wu He Xiao-Long Cui Wen-Jun Li 《Extremophiles : life under extreme conditions》2010,14(4):397-402
A novel moderately halophilic, alkaliphilic, non-motile, non-sporulating, catalase-positive, oxidase-negative, aerobic, coccus-shaped,
Gram-positive bacterium, designated strain JSM 071043T, was isolated from a subterranean brine sample collected from a salt mine in Hunan Province, China. Growth occurred with
0.5–20% (w/v) NaCl (optimum 5–10%) at pH 6.5–10.5 (optimum pH 8.5) and at 10–40°C (optimum 25–30°C). Good growth also occurred
in the presence of 0.5–20% (w/v) KCl (optimum 5–8%) or 0.5–25% (w/v) MgCl2·6H2O (optimum 5–10%). The peptidoglycan type was A4α (l-Lys–l-Ala–l-Glu) and major cell-wall sugars were tyvelose and mannose. The major cellular fatty acids were anteiso-C15:0, iso-C16:0 and anteiso-C17:0. Strain JSM 071043T contained MK-9 and MK-8 as the predominant menaquinones and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol
as the major polar lipids. The DNA G + C content was 67.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed
that strain JSM 071043T was a member of the suborder Micrococcineae, and was most closely related to Zhihengliuella halotolerans YIM 70185T (sequence similarity 98.9%) and Zhihengliuella alba YIM 90734T (98.2%), and the three strains formed a distinct branch in the phylogenetic tree. The combination of phylogenetic analysis,
DNA–DNA relatedness values, phenotypic characteristics and chemotaxonomic data supports the proposal that strain JSM 071043T represents a novel species of the genus Zhihengliuella, for which the name Z. salsuginis sp. nov. is proposed. The type strain is JSM 071043T (= DSM 21149T = KCTC 19466T). 相似文献
15.
Abundance, Diversity, and Dynamics of Viruses on Microorganisms in Activated Sludge Processes 总被引:1,自引:0,他引:1
We examined the abundance of viruses on microorganisms in activated sludge and the dynamics of their community structure.
Direct counting with epifluorescence microscopy and pulsed-field gel electrophoresis (PFGE) were applied to 20 samples from
14 full-scale wastewater treatment plants (wwtps) treating municipal, industrial, or animal wastewater. Furthermore, to observe
the dynamics of viral community structure over time, a laboratory-scale sequencing batch reactor was operated for 58 days.
The concentrations of virus particles in the wwtps, as quantified by epifluorescence microscopy, ranged from 4.2 × 107 to 3.0 × 109 mL−1. PFGE, improved by the introduction of a higher concentration of Tris–EDTA buffer in the DNA extraction step, was successfully
used to profile DNA viruses in the activated sludge. Most of the samples from different wwtps commonly had bands in the 40–70 kb
range. In the monitoring of viral DNA size distribution in the laboratory-scale reactor, some bands were observed stably throughout
the experimental period, some emerged during the operation, and others disappeared. Rapid emergence and disappearance of two
intense bands within 6 days was observed. Our data suggest that viruses—especially those associated with microorganisms—are
abundant and show dynamic behavior in activated sludge. 相似文献
16.
Hiroshi Takashima Yukiko Matsushima Yasuyuki Araki Osamu Ito Keiichi Tsukahara 《Journal of biological inorganic chemistry》2008,13(2):171-181
In order to elucidate the intramolecular photoinduced electron-transfer or energy-transfer mechanisms of the zinc myoglobin
(ZnMb) dyad and to construct a photoreaction system within a Mb–DNA complex, we newly prepared ZnMb appending an ethidium
ion (Et+). The steady-state fluorescence of ZnMb–Et+ at 600 nm and its lifetime (2.2 ns) indicate that the excited singlet state of 1(ZnMb)* is not quenched by the Et+ moiety, whereas the lifetime of the excited triplet state of 3(ZnMb)*–Et+ was shorter (τ = 4.3 ms) than those of ZnMb and the intermolecular (ZnMb + ethidium) system. Upon photoirradiation of Et+, fluorescence studies indicated the intramolecular quenching reactions from the excited singlet state, 1(Et+)*, to ZnMb, the process of which is likely the photoinduced energy-transfer reaction via a through-space mechanism. We also
demonstrate the photophysical and spectroscopic properties of ZnMb–Et+ in the presence of calf thymus (CT) DNA. The changes in the absorption and fluorescence spectra of ZnMb–Et+ on the addition of CT-DNA up to 15 equiv were very small, indicating that there are no major changes in the heme pocket.
However, we observed a longer lifetime of 3(ZnMb)*–Et+ in the presence of CT-DNA (τ = 5.3 ms) by single flash photolysis. This was induced by noncovalent interactions between Et+ and CT-DNA, followed by a conformational change of Et+ at the surface of ZnMb, where the donor–acceptor distance was probably elongated by CT-DNA. The synthetic manipulation at
the Mb surface, by using a DNA intercalator coupled with photoinduced reaction, may provide a sensitive transient signal for
DNA and valuable information to construct new Mb–DNA complex.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
17.
Copper (II) complex of formulation [Cu–Phen–Tyr](H2O)](ClO4) (Phen = 1,10-phenanthroline, l-Tyr = l-tyrosine), has been prepared, and their induced DNA oxidative cleavage activity studied. The complex binds to DNA by intercalation,
as deduced from the absorption and fluorescence spectral data. Scatchard plots constructed from the absorption titration data
gave binding constant 2.44 × 104 M−1 of base pairs. Extensive hypochromism, broadening, and red shifts in the absorption spectra were observed. Upon binding to
DNA, the fluorescence from the DNA–ethidium bromide system was efficiently quenched by the copper (II) complex. Stern–Volmer
quenching constant 0.61 × 103 M−1 obtained from the linear quenching plots. [Cu–Phen–Tyr] complex efficiently cleave the supercoiled DNA to its nicked circular
form with gallic acid as biological reductant at appropriate complex concentration. The gallic acid as reductant could observably
improve copper (II) complex to DNA damage. The pseudo-Michaelis–Menten kinetic parameters (k
cat, K
M) were calculated to be 1.32 h−1 and 5.46 × 10−5 M for [Cu–Phen–Tyr] complex. Mechanistic studies reveal the involvement of superoxide anions and hydroxyl radical (HO·) as the reactive species under an aerobic medium. 相似文献
18.
Yingmei Zhang Yejing Wang Runliu Yu Sheng Zhang Zhenbin Wu 《Frontiers of Biology in China》2008,3(1):50-54
The effects of heavy metals Cd2+, Pb2+ and Zn2+ at 0.05, 0.5 and 5.0 mg/L level and their interactions at 0.5 mg/L level on DNA damage in hepatopancreas of loach Misgurnus anguillicaudatus for 1–35 days exposure were examined by single cell gel electrophoresis (SCGE). For each test group, 20 loaches with similar
body size (5.17–7.99 g; 11.79–13.21 cm) were selected and kept in aquaria with dechlorinated water at (22±1)°C and fed a commercial
diet every 48 h. According to the percentage of damaged DNA with tail and its TL/D (tail length to diameter of nucleus) value,
the relationship between DNA damage degree and heavy metal dose and exposure time was determined. Results showed that the
percentage of damaged DNA and the TL/D value were increased with the prolonged exposure time. The highest percentage (84.85%)
of damaged DNA was shown in 5.0 mg/L Zn2+ group after 28 days exposure and the biggest TL/D value (2.50) in all treated groups after 35 days exposure. During the first
treated week, the damnification of DNA was mainly recognized as the first level, after that time, the third damaged level
was mostly observed and the percentage of damaged DNA was beyond 80%. The joint toxic effects among Cd2+, Pb2+ or Zn2+ revealed much complexity, but it generally displayed that the presence of Cd2+ could enhance the genotoxicity of Pb2+ or Zn2+. In conclusion, the results suggested that there was a significant time-and dose-depended relationship between the heavy
metal and DNA damage in hepatopancreas of loach, and SCGE could represent a useful means to evaluate the genotoxicity of environmental
contamination on aquatic organisms.
__________
Translated from Acta Hydrobiologica Sinica, 2006, 30(4): 399–403 [译自: 水生生物学报] 相似文献
19.
Three pigmented strains of halophilic archaea, RS94-RS96, were isolated from acidic foamy products of flotation enrichment of potassium minerals (Silvinit Co., Solikamsk, Russia). The cells were gram-negative, nonmotile, pleomorphic ovoids, 1.0−1.5 × 1.5−2.5 μm. The isolates were chemoorganotrophic, obligately aerobic, and catalase-positive. A range of carbohydrates and organic acids was used, as well as amino acids and peptides. The strains were halophiles and thermotolerant neutrophiles. They grew in the media with 15 to 30% NaCl (optimum at 20–22%) and 0.005–0.7 M Mg2+ (0.1–0.2 M), at pH 5.0–8.2 (optimum 7.0–7.2) and 25–55°C (optimum at 35–50°C). The major fatty acids were C16:0, C18:1, C18:0, and C16:1. The membranes contained carotenoid pigments of the bacterioruberin series and polar lipids, mostly as C20,C20 isoprenoid derivates: phosphatidylglyceromethylphosphate, phosphatidylglycerol, and three unidentified sulfated glycolipids of the S-DGD type. The DNA G+C content was 65.1–66.4 mol %. Phylogenetic analysis based on the 16S rRNA gene sequencing revealed that the thermotolerant neutrophilic isolate RS94 (DNA G+C content of 66.4 mol %) was most closely related to the nonpigmented moderate acidophile Halarchaeum acidiphilum MH1-52-1T (97.3%). Based on its phenotypic and genotypic characteristics, the organism was classified as a new species of the genus Halarchaeum with the proposed name Halarchaeum solikamskense sp. nov. The type strain is RS94T (= VKPM B-11282T). 相似文献
20.
Optimization and scale-up of a new photobleaching agar extraction process from Gracilaria lemaneiformis 总被引:1,自引:0,他引:1
Haiyan Li Jianyu Huang Yanjuan Xin Biaoming Zhang Yan Jin Wei Zhang 《Journal of applied phycology》2009,21(2):247-254
An eco-friendly photobleaching extraction process for agar extraction from the red alga Gracilaria lemaneiformis was developed for the benefit of workers’ health and environmental safety. Here we report the optimization of key process
parameters (alkali modification concentration, photobleaching duration, algal length and screen filter opening size) in order
to scale up this new technique. The optimal conditions were found to be modification by 3–5% NaOH, photobleaching for 5 h,
using algal fragments 2 –4 cm in length, and a filter screen with a 6 μm opening. A 20-L agar extraction reactor was thus
constructed, and the scale-up of the agar extraction process was tested in six batch experiments. The resulting agar quality
was similar to that of the laboratory-scale extraction. In addition, batch-to-batch reproducibility was excellent. The results
demonstrate the excellent scale-up ability and potential application of this new photobleaching agar extraction process on
a commercial scale. The agar yield and gel strength for 5% NaOH modified agar were 26.8% and 1,897 g cm−2, while those for 3% NaOH modified agar were 28.2% and 1,287 g cm−2, respectively. It is clear that the agar yield and quality can be manipulated via alkali modification in this new eco-friendly
extraction to meet market demands. 相似文献